Chromosome segregation is crucial to the health and survival of all organisms. Accumulating evidence indicates that the vast majority of human cancers display high rates of chromosome missegregation. This phenomenon, termed chromosomal instability (CIN), has been proposed to drive tumorigenesis by facilitating the emergence of aneuploid clones with increased malignant potential. Thus, it is imperative to obtain a better understanding of the molecules and mechanisms that regulate the process of chromosome segregation in human cells and sustain its high fidelity. We have developed novel methods for 'knocking out'genes in human somatic cells, and we have used these methods to characterize the role of a small mitosis-regulating protein termed securin. This protein forms a tight complex with a large cysteine protease termed separase, which digests the physical links that hold sister chromatids together. Based on our preliminary studies, we hypothesize that the securin-separase complex plays a central role in mitotic progression and high-fidelity chromosome segregation in humans. Precise regulation of this complex is proposed to link sister chromatid separation to upstream checkpoints and restrict it to the the appropriate phase of the cell cycle, i.e., anaphase. To test these hypotheses, we will investigate how specific post-translational modifications contribute to the function of the separase-securin complex. In Aim 1, we use a genetic approach to elucidate the in vivo role of separase auto-cleavage, to determine its relevance to mitotic progression and chromosome dynamics. In Aim 2, we test a proposed securin-independent mechanism for inhibiting premature sister chromatid separation, taking advantage of the hSecurin- 'knockout'cells developed in our earlier studies. In Aim 3, we evaluate potential mechanisms for separase-dependent regulation of processes other than sister chromatid separation. Together, these studies will significantly advance our understanding of mitotic chromosome segregation in humans. Such information is essential to developing more effective cancer therapies that attack the CIN phenotype found in most cancer cells.